Discovery of new viruses is often hindered by difficulties in their amplification in cell culture and/or lack of their cross-reactivity in serological and nucleic acid hybridization assays to known viruses. Many new viruses have been recently identified in environmental and clinical samples using metagenomic approaches, in which viral particles are first purified and viral nucleic acid sequences are then randomly amplified prior to subcloning and sequencing (Delwart, 2007).
The Dicer family of host immune receptors mediates antiviral immunity in fungi, plants and invertebrate animals by RNA interference (RNAi) or RNA silencing (1-3). In this immunity, a viral double-stranded RNA (dsRNA) is recognized by Dicer and diced into small interfering RNAs (siRNAs). These virus-derived siRNAs are then loaded into an RNA silencing complex to act as specificity determinants and to guide slicing of the target viral RNAs by an Argonaute protein (AGO) present in the complex. Dicer proteins typically contain an RNA helicase domain, a PAZ domain shared with AGOs, and two tandem type III endoribonuclease (RNase III) domains. Dicer cleaves dsRNA with a simple preference toward a terminus of dsRNA, producing duplex small RNA fragments of discrete sizes progressively from the terminus (4).
In addition to siRNAs, microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) also guide RNA silencing in similar complexes but with distinct AGOs (4-6). In Drosophila melanogaster, miRNAs and siRNAs are predominantly 22 and 21 nucleotides in length, dependent on Dicer-1 (DCR1) and DCR2 for their biogenesis, and act in silencing complexes containing AGO1 and AGO2 in the AGO subfamily, respectively (4-6). In contrast, ˜24-30-nt piRNAs are Dicer-independent and require AGO3, Aubergine (AUB) and PIWI in the PIWI subfamily for their biogenesis (4-6). Genetic analyses (7-10) have clearly demonstrated a role for D. melanogaster DCR2 in the immunity and biogenesis of viral siRNAs targeting diverse positive-strand (+) RNA viruses, including Flock house virus (FHV), cricket paralysis virus, Drosophila C virus (DCV), and Sindbis virus (SINV). Cloning and sequencing of small RNAs from FHV-infected Drosophila cells further indicate that the viral dsRNA replicative intermediates (vRI-dsRNA) are the substrate of DCR2 and the precursor of viral siRNAs (11-12). Drosophila susceptibility to Drosophila X virus (DXV), which contains a dsRNA genome, is influenced by components from both the siRNA (e.g., AGO2 & R2D2) and piRNA (e.g., AUB & PIWI) pathways (13). However, detection of small RNAs derived from any dsRNA virus has not been reported yet (1, 13).
Virus-derived small RNAs were first detected in plants infected with a +RNA virus (14). The Dicer proteins involved in the production of siRNAs targeting both +RNA viruses and DNA viruses have been identified in Arabidopsis thaliana (2-3), which encode AGOs in the AGO subfamily but not in the PIWI subfamily (15). Cloning and sequencing of plant viral siRNAs suggest that they may be processed either from vRI-dsRNA or hairpin regions of single-stranded RNA precursors (16-20). Production of viral siRNAs has also been demonstrated in fungi, silkworms, mosquitoes, and nematodes in response to infection with +RNA viruses and viral small silencing RNAs produced in fungi and mosquitoes have recently been cloned and sequenced (21-25).
The available data thus illustrate that accumulation of virus-derived small silencing RNAs is a common feature of an active immune response to viral infection in diverse eukaryotic host species.